19 - Direct demonstration of virus infections I

Question 1

Direct demostration of virus infections

Which methods should be mentioned?

Answer 1

1. Virus isolation
2. Diagnostic experimental animal infection
3. Electron-microscope investigation (topic 20)
4. Virus antigen detection (topic 20)
5. Viral nucleic acid demonstration (topic 20)

Question 2

Virus isolation

How is the virus isolated?

Answer 2

In vitro cultivation and identification

Question 3

Virus isolation

What is the prerequisite of in vitro propagation?

Answer 3

Infective virion → early phase (acute stadium) → virus shedding

Question 4

Virus isolation

Give the period of the diagnosis

Answer 4

2-3 weeks

Question 5

Virus isolation

Importance and significance

Answer 5

• Old, but still very important
• Not replacable
• If isolated - helps to diagnose new viruses or mutations

Question 6

Virus isolation

Where can the sample be taken from?

Answer 6

• Either:
  
  • Animal alive (buffy coat, body fluids, faeces)
  • Animal dead (organ samples)

Question 7

Virus isolation

Sample processing

Answer 7

1. Need a diseased organ (from dead animal)
2. Diluted 1:10 in physological solution
3. Homogenisation with sand in ceramic mortar
   
   1. To release ECF
4. Rinsing 2 hours
5. Bi-centrifucation
   
   1. Low speed for 10 minutes (remove sand)
   2. Supernatant - high speed for 10 minutes (spearated bacteria)
6. Filtration: 0.45 micrometer filter

Question 8

Virus isolation

Inoculation of cell-culture or embryonated egg

Answer 8

• Adsorption: To avoid CPE caused by toxins → virus attaches to the cell surface
• Suspension: dividing cells
• Co-cultures: propagation of healthy and infected cells together
• Embryonated eggs

Question 9

Virus isolation

Shell viral assay

Answer 9

• ​Viruses:
  
  • ​Herpesviruses (CMV, HSV, VZV) (especially)
  • Adenoviruses
  • Enteroviruses
  • Flaviviruses
  • Orthomyxoviruses
  • Paramyxoviruses
• Steps:
  
  • Inoculation
  • Centrifucgation
  • Incubation (6 hours)
  • Map staining

Question 10

Virus isolation

Cell culture

Answer 10

• Daily examination
• Should be CPE
  
  • If there is no CPE, either:
    
    • Blind-passage (increase of virus titer and CPE)
    • Auxiliary examinations: EM, HA, IF, IP
• Isolate → plaque-isolation → purification → virus strain

Question 11

Virus isolation

Embryonated egg

Answer 11

• Daily examination
• Sample collection:
  
  • Allantoic fluid
  • CAM
  • Embryo
• Auxillary examinations: HA, EM, histopathology

Question 12

Virus identification

What is investigated

Answer 12

The isolate (in order to save time)

Question 13

Virus idenification

Which tests can be performed to find the virus family?

Answer 13

• To find the virus family, CPE, cell spectrum is not sufficient (riktig?)

1. Virus neutralization
   
   1. ​Serotype of species → serological methods of virus neutralization
2. Physio-chemical tests
   
   1. ​Chloroform resistance: enveloped→← non-enveloped
   2. Halogenic uridine derivated: DNA inhibitors
   3. Acridin orange staining: ssNA →← dsNA
   4. Electron microscopes: ultrathin layer / negative contrast staining

Question 14

Virus identification

Which tests can be performed to find the sereotype ≈ species?

Answer 14

Serological methods VN

Question 15

Virus identification

Which tests can be performed to find the subtype, variants?

Answer 15

• Alterations in genotype:
  
  • NA examination
    
    • REA
    • OFP
    • HD
• Alterations in structure proteins:
  
  • MAB
  • PAGE

Question 16

Diagnostic experimental animal infection

Answer 16

• In vivo prerequisite of virus propagation infective virion → acute phase → mass virus shedding
• Period of diagnosis: 8-10 days
  
  • It is faster and more reliable than virus isolation
• Can for example pe performed on:
  
  • African horse sickness
  • Bluetonuge
  • Rabies
  • FMD
  • Classical swine fever
  • African swine fever
• Auxiliary examinations:
  
  • Pathology
  • Histopathology
  • Immuno flourescence (IF)
  • Immuno peroxydase (IP)
  • Antigen ELISA